Highly selective mitochondria-targeting amphiphilic silicon(IV) phthalocyanines with axially ligated rhodamine B for photodynamic therapy

Article abstract of DOI:10.1021/ic201178e

Two axially ligated rhodamine-Si(IV)-phthalocyanine (Rh-SiPc) conjugates, bearing one and two rhodamine B, were synthesized and their linear and two-photon photophysical, subcellular localization and photocytotoxic properties were studied. These Rh-SiPc c

Full text of DOI:10.1021/ic201178e

Article
pubs.acs.org/IC
Highly Selective Mitochondria-Targeting Amphiphilic Silicon(IV)
Phthalocyanines with Axially Ligated Rhodamine B for
Photodynamic Therapy
Zhixin Zhao,† Pui-Shan Chan,‡ Hongguang Li,† Ka-Leung Wong,† Ricky Ngok Shun Wong,‡
Nai-Ki Mak,*,‡ Jie Zhang,† Hoi-Lam Tam,§ Wai-Yeung Wong,†,∥ Daniel W. J. Kwong,*,†
and Wai-Kwok Wong*,†,∥
†Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR
‡Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR
§Department of Physics, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR
∥Institute of Molecular Functional Materials, Areas of Excellence Schemes, University Grants Committee, Hong Kong
S
* Supporting Information
ABSTRACT: Two axially ligated rhodamine−Si(IV)−phthalocyanine (Rh-
SiPc) conjugates, bearing one and two rhodamine B, were synthesized and
their linear and two-photon photophysical, subcellular localization and
photocytotoxic properties were studied. These Rh-SiPc conjugates exhibited
an almost exclusive mitochondrial localizing property in human nasophar-
yngeal carcinoma (HK-1) cells and human cervical carcinoma (HeLa) cells.
Strong photocytotoxic but low dark cytotoxic properties were also observed
for the two Rh-SiPc conjugates toward the HK-1 cells. Using nuclei staining
method and flow cytometric DNA content analysis, apoptotic cell death was
induced by these conjugates upon photoactivation. This observation is
consistent with their mitochondrial localization property. The observed
properties of these conjugates qualify them as promising PDT agents.
inflammatory responses often seen in conventional chemotherapy.5
In this work, we report the synthesis of a mitochondria-targeting
INTRODUCTION
Photodynamic therapy (PDT) has attracted increasing
■
PDT agent by covalently connecting a well-known mitochondria-
attention as a highly selective anticancer treatment modality
targeting agent, namely, rhodamine B (Rh B), to a tumor-
where a nontoxic photosensitizer showing preferential accu-
mulation in tumor tissue or its vasculature when administered
localizing photosensitizer, namely, phthalocyanine (Pc), and
evaluated its subcellular localization, one- and two-photon PDT
inside the body is activated by the tumor-targeted photo-
properties in several cancer and noncancer cell models.
irradiation at a specific wavelength to produce cytotoxic reactive
oxygen species (ROS), principally singlet oxygen (1O2), which
This design is based on the fact that positively charged rho-
damine, such as Rh B and rhodamine 123,6 accumulates speci-
fically in the mitochondria of living cells and has been used as
probes to measure and monitor mitochondrial membrane
potential in bioassays.7 On the other hand, Pc is a class of
photosensitizers that absorbs strongly in the tissue-penetrating
spectral window of 650−800 nm and has been extensively
studied as a potential PDT agent due to its tumor-localizing
properties.3,8 Since most phthalocyanines are insoluble and
tend to aggregate in aqueous media, they have to be encap-
sulated in liposomes,9 polymeric micelles10 or nanoparticles11
before their administration into test animals or cells. The
aqueous solubility of Pc can be improved by introduction of
hydrophilic substituents, such as sulfonate,12 carboxylate,13
N-methylpyridinium,14 and polyethylene glycol (PEG),15 in the
then kills the tumor cells.1,2 An ideal anticancer PDT agent
should possess the following properties: (1) minimal dark cyto-
toxicity, (2) relatively high O2 quantum yield, (3) preferential
uptake by target tissues, (4) strong absorption at longer wave-
lengths, that is, λ > 650 nm, where absorption by biomolecules,
such as hemoglobin, is minimal, (5) high chemical stability, and
(6) rapid clearance from normal tissues.3 As singlet oxygen is
believed to be the principal cytotoxic agent, its short reactive
range (0.01−0.02 μm) in biological systems4 is an important
factor to consider in designing an efficacious PDT agent. To
1
1
overcome the short reactive ranges of O2 and other ROS,
photosensitizer needs to closely localize to the targets that can
effectively trigger cell death. Among the intracellular targets
that have been shown to cause cell death, mitochondrion is
considered quite appealing as it can cause cell death by apoptosis,
which is a programmed shutdown of the cell machinery with
all its components recycled without eliciting any undesirable
Received: May 31, 2011
Published: December 22, 2011
© 2011 American Chemical Society
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dye E12353 (Invitrogen) for 30 min. The emitted fluorescent signals
of 2, 3, Rh B, 4 and the organelle-specific probes were examined using
the (Olympus) FV1000 confocal microscope equipped with a spectral
detection system. A diode laser line at 405 nm was used for excitation
of the ER Tracker. An argon-ion laser line at 488 nm was used for
the excitation of Mito Tracker, Lyso Tracker and Golgi Tracker. A
helium−neon laser line at 543 nm was used for the excitation of 2, 3,
Rh B and 4. Emission signals at 425−475 nm (ER Tracker), 500−
530 nm (Mito, Lyso and Golgi Trackers), 620−720 nm (2 and 3),
560−660 nm (Rh B) and 630−710 nm (4) were collected. A 60x oil
immersion objective and pinhole size of 110 μm was used for image
capturing. Images were processed and analyzed using the FV10-ASW
software (Olympus).
macrocyclic ring. Axial ligation of hydrophilic or amphiphilic
groups in metallophthalocyanines, including silicon(IV)
phthalocyanine (SiPc), which is currently under clinical trial,8
is another approach to increase water solubility and reduce ag-
gregation in aqueous media.16 Recently, the synthesis and PDT
activity of the hydrophobic monohydroxy trisphenylporphyrin
(TPP-OH) tethered to Rh B via a saturated hydrocarbon linker
has been reported.17 Enhanced (>5-fold) photocytotoxicity,
relative to its components (i.e., TPP-OH and Rh B), and pre-
ferential accumulation at mitochondria were observed for this
conjugate. Similar conjugates of sulfono- and carboxy-substituted
phthalocyanines with rhodamines have also been synthesized18
and their efficient intramolecular energy transfer (from Rh to Pc)
studied as well.19 In our design, Rh B, instead of connecting to
the macrocyclic ring as peripheral substituents as in previous
works,17−19 is axially connected to SiPc via a polyether linkage
to form two SiPc-Rh B conjugates, 3 and 2, possessing one and
two Rh B moieties, respectively. This axial ligation design can
reduce Pc aggregation, thereby increasing its excited state
lifetimes20 and improving its cellular uptake and imaging pro-
perties. The synthesis, linear and two-photon photophysical,
subcellular localization, and in vitro PDT activity of these con-
jugates are reported herein.
Photocytotoxicity Assay. HK-1 cells (2 × 104/well) were
incubated in wells of 96-well plate for overnight. The cells were
treated with 2 (100 nM), 3 (100 nM), Rh B (100 nM), or 4 (100 nM)
for 6 h in dark. The culture medium was then replaced with fresh
medium and the cells were exposed to yellow light (1−4 J/cm2)
produced from a 400 W tungsten lamp fitted with a heat-isolation filter
and a 500 nm long-pass filter. The fluence rate was 4 mW/cm2. Cells
viability was determined by the MTT reduction assay at 24 h post-
PDT.22 The cell monolayers were rinsed twice with phosphate-
buffered saline (PBS) and then incubated with 250 μg/mL MTT
solution at 37 °C for 3 h. The formazan crystal formed was dissolved
in DMSO and the absorbance of dissolved formazan crystal at 540 and
690 nm was measured using a 96-well plate reader (ELx800
Absorbance Microplate Reader).
EXPERIMENTAL SECTION
■
General Remarks. All reactions were carried out in a dry nitrogen
atmosphere. Solvents were dried by standard procedures, distilled, and
deaerated prior to use. All chemicals were obtained from Aldrich
Chemical Co. and, where appropriate, degassed before use. NMR
spectra were recorded with a Varian INOVA 400 NMR spectrometer.
High-resolution matrix assisted laser desorption/ionization time-of-
flight (MALDI-TOF) mass spectra were recorded with a Bruker Autoflex
MALDI-TOF mass spectrometer. Electronic absorption spectra in the
UV/vis region were recorded with a Varian Cary 100 UV/vis spec-
trophotometer. Steady-state visible fluorescence and photolumines-
cence excitation spectra were recorded with a Photon Technology
International (PTI) Alphascan spectrofluorimeter and singlet-oxygen
near-IR (NIR) emission spectra were recorded directly by its pho-
sphorescence emission at 1270 nm with an InGaAs detector, using
H2TPP (ΦΔ = 0.55 0.11)21 as the reference compound. The filter
was LG-697-F from Corion Company. Infrared spectra (KBr pellets)
were recorded with a Nicolet Magna 550 FTIR spectrometer. All
measurements were performed at ambient temperature (20 2 °C)
and pressure. The UV/vis absorption and NIR emission spectra of all
solution samples were measured in a 10 mm quartz cell.
[(absorbance of cell control − absorbance of blank)
− (absorbance of treatment − absorbance of blank)]
/(absorbance of cell control − absorbance of blank)
× 100%
DNA Content Analysis. HK-1 cells (3 × 105/well) were seeded
onto 35 mm Petri dish for overnight. The cells were treated with 2
(100 nM) for 6 h in dark. The cells were then irradiated (1−4 J/cm2)
as described above. At 24 h post-PDT, both floating and adherent cells
were collected, washed with PBS twice and then fixed in 70% ethanol
for at least 1 h at 4 °C. After fixation, the cells were stained with DNA-
binding buffer (40 μg/mL propidium iodide, 1 mg/mL RNase, 0.1%
Triton X-100 in PBS) for 30 min. The fluorescence profiles of the
stained cells were analyzed using the FACSCalibur Flow Cytometer
(Becton−Dickinson).23 Laser with a wavelength of 488 nm was used
for excitation, and the fluorescence signal was detected using the FL-2
channel (λem = 564−606 nm). At least 10 000 events were counted.
DNA content was analyzed using the Cell Quest and the Modfit LT
Version 3.0 Software.
Cell Culture. Human nasopharyngeal carcinoma (HK-1) cells
were cultured in RPMI-1640 medium (Gibco) supplemented with
10% fetal bovine serum (Gibco) and antibiotics (penicillin 50 U/mL;
streptomycin 50 μg/mL). Human cervical carcinoma (HeLa) cells
were maintained in an RMPI 1640 medium supplemented with 10%
fetal bovine serum (FBS) and 1% penicillin and streptomycin. Cells
were incubated at 37 °C in a humidified incubator with 5% CO2.
Culture medium in each dish was changed prior to exposure to the test
compounds. Stock solutions of the test compounds (1 mM) were
prepared in DMSO and stored in dark at room temperature. These
compounds, when used in the imaging and bioassay experiments
involving cultured cells, were diluted with the corresponding culture
media to appropriate concentrations.
Confocal Microscopic Imaging of Rh-SiPc Conjugates. HK-1
cells (1 × 105) were seeded onto coverslip in 35-mm culture dishes
for overnight. The cells were initially incubated with Rh-SiPc-Rh
(2, 50 nM), Rh-SiPc (3, 50 nM), Rh B (1 μM) or C-SiPc-C7 (4, 1 μM)
for 30 min in dark. The cells were then washed and stained with 100 nM
mitochondria-specific probe, Mito Tracker Green FM dye M7514,
lysosomes-specific probe Lyso Tracker Green DND-26 L7526, Golgi-
specific probe BODIPY FL C5-ceramide complexed to BSA B-22656
and the endoplasmic reticulum probe ER Tracker Blue−White DPX
Staining of Apoptotic Nuclei. Hoechst 33258 was used in the
staining of apoptotic nuclei.24 Overnight cultured HK-1 cells were treated
with 2 (100 nM) for 6 h in dark and then irradiated (1−4 J/cm2)
as described previously. At 3 and 16 h after PDT, detached and
adherent cells were collected and fixed with 4% paraformaldehyde in
PBS for 15 min. Cell membrane was permeabilized with absolute
methanol for 20 min, followed by staining with Hoechst 33258
(0.12 μg/mL) in PBS for 15 min. Fluorescence images of apoptotic
nuclei were visualized and captured under a fluorescence microscope
(Zeiss, Axioskop2).
Two-Photon-Induced Confocal Microscopic Imaging of
Rh-SiPc Conjugates. HeLa cells (1 × 105) were seeded onto cover-
slip in 35-mm culture dishes for overnight. The cells were initially
incubated with Rh-SiPc-Rh (2, 5 μM), Rh-SiPc (3, 5 μM), or C7-SiPc-
C7 (4, 5 μM) for 6 h in dark. The two-photon-induced fluorescent
signals of 2, 3, and 4 were captured using the Leica SP5 (upright
configuration) confocal microscope equipped with a femtosecond-
pulsed Ti:Sapphire laser (Libra II, Coherent) inside the tissue culture
chamber (5% CO2, 37 °C). The excitation beam produced by the
femtosecond laser, which was tunable from 680 to 1050 nm, was
focused on the adherent cells through a 40× oil immersion objective.
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Article
The time-lapse images (0−30 min, one laser shot per min) were obtained
with femtosecond laser excitation at 850 nm (laser power ∼8 mW).
Two-Photon Absorption Measurement. Two-photon absorp-
tion spectra of 2, 3, and 4 were measured at 850 nm by the open-
aperture Z-scan method25 using 100-fs laser pulses with a peak power
of 276 GW cm−2 from an optical parametric amplifier operating at a
1-kHz repetition rate generated from a Ti:Sapphire regenerative
amplifier system. The laser beam was split into two parts by a beam
splitter. One was monitored by a photodiode (D1) as the incident
intensity reference, I0, and the other beam was detected by the
photodiode (D2) as the transmitted intensity. After passing through a
lens with f = 20 cm, the laser beam was focused and passed through a
quartz cell. The position of the sample cell, z, was moved along the
laser-beam direction (z axis) by a computer-controlled translatable
table so that the local power density within the sample cell could be
changed under the constant incident intensity laser power level.
Finally, the transmitted intensity from the sample cell was detected by
the photodiode D2 interfaced to a computer for signal acquisition and
averaging. Each transmitted intensity data represent an average of over
100 measurements. Assuming a Gaussian beam profile, the nonlinear
absorption coefficient β can be obtained by curve fitting to the
observed open-aperture traces, T(z), with eq 1,
(d, J = 7.6 Hz, 1H; Har), 7.77 (m, 2H; Har), 7.24 (d, J = 7.6 Hz, 1H;
Har), 7.02 (d, J = 9.6 Hz, 2H; Har), 6.78 (d, J = 9.6 Hz, 2H; Har), 6.72
(s, 2H; Har), 4.08 (t, J = 4.7 Hz, 2H; OCH2), 3.52 (m, 8H;
NCH2CH3), 3.45 (m, 14H; OCH2), 2.56 (broad s, 1H; OH), 1.25 (t,
J = 7.1 Hz, 12H; NCH2CH3) ppm; 13C NMR (100 MHz, CD2Cl2) δ =
165.3, 159.5, 158.2, 155.9, 134.0, 133.4, 131.8, 131.7, 130.8, 130.5,
130.2, 114.5, 113.9, 96.5, 72.9, 70.9,70.8, 70.7, 70.6, 69.0, 65.1, 61.8,
46.5, 12.8 ppm; HRMS (MALDI) m/z calcd for [C36H47N2O7]+
+
619.3378 [M − Cl] ; found 619.3374; Δm = −0.65 ppm.
Synthesis of Rh-SiPc-Rh (2) and Rh-SiPc (3). A mixture of
tetraethylene glycol rhodamine B (1, 123.8 mg 0.2 mmol), SiPcCl2
(61.2 mg, 0.1 mmol) and 0.5 mL dried pyridine in dried toluene
(20 mL) was refluxed for 24 h. After cooling to room temperature, the
solvent, which contained the unreacted 1 and the byproduct SiPc-
tetraethylene glycol without the Rh B, was poured out. The sticky blue
liquid mixture adhered to the bottom of the reaction flask was
dissolved in a minimum volume of CH2Cl2 and loaded onto a basic
Al2O3 column. Product 3 was eluted with CH2Cl2/MeOH (80:20) as a
blue band and product 2 was eluted with CH2Cl2/MeOH (75:25) as a
purple-blue band. Removal of solvent by passage of air stream afforded
the purplish blue solid product 3 (7.76 mg, 4.2%) and the deep blue-
pale purple colored solid product 2 (19.4 mg, 10.5%). Both products
were purified by recrystallization from CH2Cl2/Et2O. 2: UV/vis
[CH2Cl2, λmax/nm (log ε)] 353 (4.63), 558 (4.89), 607 (4.13), 645
(4.09), 675 (4.93); 1H NMR (400 MHz, CD2Cl2) δ = 9.54 (d, J = 2.7
Hz, 8H; Pc-1,4-Har), 8.29 (d, J = 2.7 Hz, 8H; Pc-2,3-Har), 8.04 (d, J =
7.6 Hz, 2H; Rh-Har), 7.63 (t, J = 7.6 Hz, 2H; Rh-Har), 7.49 (t, J = 7.8
Hz, 2H; Rh-Har), 7.20 (d, J = 7.8 Hz, 2H; Rh-Har), 6.86 (d, J = 9.4 Hz,
4H; Rh-Har), 6.62 (d, J = 9.4 Hz, 4H; Rh-Har), 6.54 (s, 4H; Rh-Har),
3.81 (t, J = 4.5 Hz, 4H; OCH2), 3.45 (m, 16H; NCH2CH3), 3.06 (t,
J = 4.5 Hz, 4H; OCH2), 2.91 (t, J = 4.7 Hz, 4H; OCH2), 2.75 (t, J =
4.7 Hz, 4H; OCH2), 2.30 (t, J = 4.7 Hz, 4H; OCH2), 1.58 (t, J = 4.7
Hz, 4H; OCH2), 1.12 (m, 24H; NCH2CH3), 0.27 (t, J = 5.6 Hz, 4H;
Si-OCH2CH2O), −2.05 ppm (t, J = 5.6 Hz, 4H; Si-OCH2CH2O);
HRMS (MALDI) m/z calcd for [C104H108N12O12Si]2+ 1777.7901
[M − 2Cl]+; found 1777.7910; Δm = 0.46 ppm. 3: UV/vis [CH2Cl2,
λmax/nm (log ε)] 343 (4.57), 559 (4.73), 611 (4.24), 649 (4.16), 679
−α S
0
βI (1 − e
)
0
T(z) = 1 −
2
2a [1 + (z/z )]
(1)
0
0
where a0 is the linear absorption coefficient, l is the sample length
(1 mm quartz cell) and z0 is the diffraction length of the incident beam.
After the nonlinear absorption coefficient β was obtained, the two-
photon absorption cross-section σ2 of the sample molecule (in units
of GM, where 1 GM = 10−50 cm4 s photon−1) can be calculated using
eq 2
1000βhv
σ =
2
N d
(2)
A
where NA is the Avogadro constant, d is the concentration of the
sample compound in solution, h is the Planck constant, and v is the
frequency of the incident laser beam.
Singlet Oxygen Quantum Yield Measurement. Singlet oxygen
was detected directly by its phosphorescence emission at 1270 nm
using an InGaAs detector on a PTI QM4 luminescence spectrometer.
The singlet oxygen quantum yields (ΦΔ) of the test compounds were
determined in CHCl3 by comparing the singlet oxygen emission
intensity of the sample solution to that of a reference compound
(H2TPP, ΦΔ = 0.55 in CHCl3)26 according to eq 327
1
(4.99); H NMR (400 MHz, CD2Cl2) δ = 9.49 (m, 8H; Pc-1,4-Har),
8.23 (d, J = 7.6 Hz, 1H; Rh-Har), 8.18 (m, 8H; Pc-2,3-Har), 7.67 (m,
2H; Rh-Har), 7.30 (d, J = 7.6 Hz, 1H; Rh-Har), 6.92 (d, J = 9.4 Hz, 2H;
Rh-Har), 6.52 (d, J = 9.4 Hz, 2H; Rh-Har), 6.34 (s, 2H; Rh-Har), 4.01
(t, J = 4.6 Hz, 2H; OCH2), 3.20 (m, 10H; OCH2 + NCH2CH3), 2.95
(t, J = 4.6 Hz, 2H; OCH2), 2.69 (t, J = 4.6 Hz, 2H; OCH2), 2.25 (t, J =
4.8 Hz, 2H; OCH2), 1.56 (t, J = 4.8 Hz, 2H; OCH2), 1.00 (t, J = 7.2
Hz, 12H; NCH2CH3), 0.29 (t, J = 5.4 Hz, 2H; SiOCH2CH2O), −2.00
(t, J = 5.40 Hz, 2H; SiOCH2CH2O) ppm; HRMS (MALDI) m/z calcd
for [C68H62N10O8Si]+ 1175.4594 [M − Cl]+; found 1175.4604; Δm
0.85 ppm.
=
2
S
⎛
⎞
G
n
A
REF
REF
S
Δ
S
Φ
= Φ
×
×
⎜
⎝
⎟
Δ
Δ
REF
Synthesis of C7-SiPc-C7 (4). A mixture of tri(ethyl glycol)-
monomethylether (79 μL, 0.5 mmol), SiPcCl2 (30.5 mg, 0.05 mmol),
and anhydrous K2CO3 (27.6 mg, 0.2 mmol) in dried toluene (10 mL)
was refluxed for 14 h. After it was cooled to room temperature, the
mixture was directly loaded onto a basic Al2O3 column. Product 4 was
eluted with CH2Cl2/MeOH (80:5) as a green band. Removal of
solvent by evaporation afforded a green solid product 4 (6.7 mg,
15.6%). The crude product was purified by recrystallization from
CH2Cl2/Et2O: UV/vis [CH2Cl2, λmax/nm (log ε)] 354 (4.01), 605
(3.80), 643 (3.72), 673 (4.62); 1H NMR (400 MHz, CD2Cl2) δ = 9.63
(m, 8H; Pc-1,4-Har), 8.34 (m, 8H; Pc-2,3-Har), 3.14 (s, 6H; OCH3),
3.12 (t, J = 4.6 Hz, 4H; OCH2), 2.95 (t, J = 4.8 Hz, 4H; OCH2), 2.45
(t, J = 4.9 Hz, 4H; OCH2), 1.67 (t, J = 4.9 Hz, 4H; OCH2), 0.38 (t, J =
5.7 Hz, 4H; SiOCH2CH2O), −1.92 (t, J = 5.7 Hz, 4H; SiOCH2CH2O)
ppm; 13C NMR (100 MHz, CD2Cl2) δ = 29.70, 54.68, 58.79, 68.56,
69.30, 69.70, 71.46, 123.64, 130.83, 135.98, 149.21 ppm; HRMS
(MALDI) m/z calcd for [C46H46N8O8SiNa]+ 889.3100 [M + Na]+;
found 889.3149; Δm = 5.51 ppm.
n
A
⎠
G
REF
S
(3)
Δ
where ΦΔ is the singlet oxygen quantum yield, GΔ is the integrated
emission intensity, A is the absorbance at the excitation wavelength, n
is the refractive index of the solvent. Superscripts REF and S cor-
respond to the reference and the sample, respectively. In all measu-
1
rements, the O2 emission spectra were obtained using an excitation
with the absorbance set at 0.05 in order to minimize reabsorption of
the emitted light.
Synthesis of Tetraethylene Glycol Rhodamine B, 1. A mix-
ture of tetraethylene glycol monoiodide (912 mg, 3 mmol), rhodamine
B (1.437 g, 3 mmol) and K2CO3 (415 mg, 3 mmol) in DMF (50 mL)
was heated at 70 °C for three days. The product mixture was filtered
and the filtrate was evaporated under vacuum. The solid obtained
was dissolved in a minimum amount of CH2Cl2 and then loaded onto
a silica gel column. The first purple-red band, which corresponded
to the unreacted rhodamine B, was eluted with EtOAc/MeOH/
Et3N (85:10:5). The second purple-red band, which corresponded
to product 1, was eluted with CH2Cl2/MeOH (75/25). Removal of
solvent by evaporation afforded the purple-brown color sticky pro-
duct 1 (987 mg, 53.1%): UV/vis [CH2Cl2, λmax/nm (log ε)] 521
(3.94), 557 (4.66); 1H NMR (400 MHz, CD2Cl2): δ = 8.28
X-ray Crystallography. X-ray diffraction data were collected at
173 K using graphite-monochromated Mo Kα radiation (λ = 0.71073
Å) on a Bruker Axs SMART 1000 CCD diffractometer. The collected
frames were processed with the software SAINT+28 and an absorption
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Scheme 1. Synthesis Route to the Si(IV)Pc-Rhodamine B Conjugates, 2 and 3, and the Si(IV)Pc with Axially Ligated
Polyethylene Glycol, 4
correction (SADABS)29 was applied to the collected reflections. The
phthalocyanine-rhodamine B conjugates, 2 and 3, is shown in
Scheme 1. Briefly, tetraethylene glycol monoiodide was pre-
pared according to the literature procedure.31 Treatment of the
commercially available Rh B with tetraethylene glycol mono-
iodide in the presence of K2CO3 in DMF gave the tetraethylene
glycol-rhodamine B, 1, in 53% yield after its separation from the
unreacted Rh B by silica gel column chromatography. Further
washing of the silica gel column with the eluting solvent allowed a
second harvest of 1, resulting in a combined yield of 70%.
Products 2 and 3 were synthesized according to the literature
procedure.32 Bases, such as K2CO3, NaH, quinoline, DMF,
pyridine, have been used to render silicon(IV) phthalocyanine
dichloride (SiPcCl2) reactive to hydroxyl derivatives. In our
preparation of 2 and 3, K2CO3 and pyridine were tried. Both
bases gave a number of byproduct of SiPc containing one and
structure was solved by the Direct method (SHELXTL)30 in
conjunction with standard difference Fourier techniques and
subsequently refined by full-matrix least-squares analyses on F2.
Hydrogen atoms were generated in their idealized positions and all
non-hydrogen atoms were refined anisotropically. Crystal data for 1:
C H N O Si; M = 867.00; triclinic; space group P1; a = 7.9377(8),
̅
46 46
8
8
b = 10.210(1), c = 13.216(1) Å; α = 94.452(2), β = 94.984(2), γ =
105.604(2)°; V = 1022.1(2) Å3; T = 173(2) K; Z = 1; reflections
collected/unique, 6493/4666, Rint = 0.0184; final R indices [I > 2σ(I)],
R1 = 0.0366, R2 = 0.0969 for 4162 observed reflections; R indices (all
data), R1 = 0.0415, R2 = 0.1013; GOF = 1.040; CCDC 779323.
RESULTS AND DISCUSSION
Synthesis of Silicon(IV) Phthalocyanine-Rhodamine B
Conjugates. The synthetic route for the two silicon(IV)
■
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Figure 1. Perspective drawing of 4, with the thermal ellipsoids drawn at the 25% probability level. Selected bond lengths (Å) and angles (deg):
Si−N(1) 1.920(1), Si−N(3) 1.9333(9), Si−O(1) 1.6971(8); N(1)−Si−N(3) 89.88(4), N(1)−Si−O(1) 86.61(4).
two polyethers without the Rh B. These byproduct were iden-
tified after TLC separation by MALDI-TOF-MS and NMR.
The yields of 2 and 3 were found to be similar for both bases.
However, with pyridine as base, products 2 and 3 were found in
the sticky liquid adhered to the bottom of the reaction flask,
while the byproduct and the starting material, 1, were present
in the toluene solvent bulk and were therefore removed readily.
Products 2 and 3 were then isolated and purified by basic
alumina column chromatography. Dichloromethane/methanol
was used as the eluting solvent to elute products 2 (yield
10.5%) and 3 (yield 4.2%) from the basic alumina column.
Since methanol has been reported to replace the axial ligand of
SiPc,32,33 other mixed solvents without methanol, such as
CH2Cl2/acetonitrile, CH2Cl2/tetrahydrofuran (THF), and
ethyl acetate/THF, were tried as eluent but to no avail. This
observation suggests that the cations present in 2 and 3 might
have increased their adsorption to the basic alumina column,
thus reducing the recoverable yields. Another reason for the
low observed yield could be the result of the displacement of
the axially ligated Rh B by the methanol used in elution. In
contrast, product 4 is more stable and no obvious decom-
position was observed during purification. Both 2 and 3 are
soluble in most organic solvents, such as CH2Cl2, CHCl3, meth-
anol, acetonitrile, THF, dimethylformamide, and dimethylsulf-
oxide, and they even showed slight solubility in water, giving
purple red solutions.
δ −1.91 to 3.12 for 4. The methoxy protons of 4 appeared as a
sharp singlet at δ 3.14 ppm. High resolution mass spectra were
also measured to confirm the identity of these compounds.
Molecular ion peak at m/z 1777.7910 observed for 2 is in good
agreement with the calculated m/z value of 1777.7901 for the
[M−2Cl]+ ion of 2 (Supporting Information Figure S4). For 3,
two major molecular ion peaks were seen at m/z 1175.4604
and m/z 1158.4113, corresponding to its [M − Cl]+ and
[M − Cl − OH]+ ions, respectively (Supporting Information
Figure S6). Three major molecular ion peaks were observed
for 4, that is, at m/z 889.3149, m/z 728.3082 and m/z 703.2029,
which corresponded to its [M+Na]+ ion, [M−tri(ethyl glycol)-
monomethylether+Na]+ ion and [M−tri(ethyl glycol)-
monomethyl ether]+ ion, respectively (Supporting Information
Figure S8). The molecular structure of 4 was also confirmed by
X-ray crystallography (see Figure 1). The asymmetric unit
consists of half of a molecule and the two halves are related by a
center of inversion at the Si atom. The Si center is six-coordinate
with four Pc nitrogen donor atoms and two axial oxygen donors
from the polyethylene glycol units arranged in an octahedral
environment.
Photophysical Properties of 2 and 3. The UV/vis
spectra of 2 and 3, together with those of 1 (Rh) and 4
All products were characterized by NMR and high-resolution
mass spectrometry. Compared to the 1H NMR spectrum of Rh
B, product 1 showed two additional peaks at δ 3.56 (14 H) and
4.08 ppm (2H), corresponding to the ethylene protons of the
tetra(ethylene glycol) that are distal and proximal, respectively,
to the Rh B (Supporting Information Figure S1). The eight
new carbon peaks found at δ 62−73 ppm in the 13C NMR
spectrum of 1 are due to the eight carbon atoms of the
tetra(ethylene glycol). MALDI-TOF mass spectrum of 1 gave a
peak at m/z of 619.3374, which compared favorably (Δm
=
−0.65 ppm) to the calculated m/z of 619.3378 for the [M −
Cl]+ ion of 1 (Supporting Information Figure S2)
1
The H NMR spectra of 2, 3, and 4 showed two downfield
groups of peaks (δ = 8−10 ppm) typical of the α- and β-
protons of the Pc ring (Supporting Information Figures S3, S5,
and S7). Because of the strong ring current effect of the Pc, the
signals of the ethylene glycol protons of 2, 3, and 4 are upfield
shifted when compared to 1. These ethylene glycol signals ap-
peared as eight four-proton triplets from δ −2.05 to 3.81 ppm
for 2; seven two-proton triplets from δ −2.00 to 4.01 ppm and
another two-proton signal overlapped with Rh B methylene
group at δ 3.20 ppm for 3; and six four-proton triplets from
Figure 2. UV/vis spectra of the 1 (Rh, ε520nm = 46600; ε557nm
145000), 2 (Rh-SiPc-Rh, ε520nm = 26400; ε557nm = 5990; ε607nm
46600; ε673nm = 50000), 3 (SiPc-Rh (ε558nm = 28700; ε607nm = 16490;
ε639nm = 21000; ε672nm = 74000), and 4 (C7-SiPc-C7, ε672nm = 16400)
at concentration of ∼1 μM.
=
=
(C7-SiPc-C7) in CH2Cl2 are shown in Figure 2. The simultaneous
appearance of the rhodamine and SiPc characteristic absorptions
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in the spectra of 2 and 3 confirms their rhodamine-SiPc conjugate
structure. Furthermore, the absorption intensity of 2 at 557 nm
(rhodamine absorption peak) is ∼2-fold to that of 3. This
observation is consistent with the ligation of two and one Rh B
moiety in 2 and 3, respectively. Figure 3 shows the absorption and
emission spectra of 2 as a function of its concentration in
CH2Cl2. The spectra exhibit typical features of nonaggregated
phthalocyanines. The inset in Figure 3a shows a plot of the
absorption intensities of the SiPc Q-band and rhodamine versus
the concentration of 2. The inset in Figure 3b shows a plot of
the emissions of 2 due to its Rh B (560 nm) and SiPc (681 nm)
moieties versus its concentration. These linear plots observed in
both the absorption and emission spectra of 2 shows that this
compound is essentially free from aggregation in the indicated
concentration ranges in CH2Cl2.
The fluorescence spectra of 2 and 3 are shown in Figure 4.
For both conjugates, excitation of the rhodamine band at 515
nm led to emissions from both the Rh and the Pc moieties
while excitation of the Pc chromophore at 640 nm resulted in
emission exclusively from the Pc moiety. Furthermore, the
emission from the Pc moiety excited at 515 nm coincided with
the emission obtained from excitation at 640 nm. These obser-
vations indicate an intramolecular energy transfer from the
excited Rh B to the Pc moiety in 2 (λem = 681 nm) and 3
(λem = 688 nm), very similar to that observed previously for
Zn(II)Pc with peripherally substituted rhodamines.19 These
results clearly indicate that such intramolecular energy transfer
from Rh to Pc can occur readily with either axially ligated or
peripherally substituted rhodamines.
Recently, phthalocyanines (H2Pc and ZnPc) peripherally
substituted by four tetraphenylporphyrins (H2TPP and
ZnTPP) via alkyne linkages in a windmill fashion was
synthesized and the intramolecular porphyrin-to-Pc energy
transfer studied.36 Compared to the almost quantitative TPP-
to-Pc energy transfer, the efficiency of Rh-to-Pc energy transfer
in 2 and 3 is significantly lower. The difference is presumably
due to the long and nonconjugative alkoxy linkage between the
donor and acceptor in 2 and 3 as compared to the short and
conjugative alkyne linkage between TPP and Pc.
Production of Singlet Oxygen from 2 and 3. To assess
1
their potential as effective PDT agents, the O2 production
Figure 3. (a) UV/vis spectra of different concentrations of 2 in
CH2Cl2. The inset shows a plot of the absorbance of 2 at 557 nm
(square), due to the Rh B moiety, and at 672 nm (triangle), due to the
SiPc moiety, as a function of its concentration. (b) Emission spectra of
different concentrations of 2 in CH2Cl2. The inset shows a plot of the
emission of 2 at 560 nm (square), due to the Rh B moiety, at 681 nm
(circle) and at 753 nm (triangle), due to the SiPc moiety, as a function
of its concentration.
capabilities of these Rh-SiPc conjugates were measured based
1
on the phosphorescence intensities of the O2 produced upon
photoirradiation of these compounds. Figure 5a shows the 1O2
phosphorescence spectra of 2, 3, and H2TPP in CHCl3. Using
1
the O2 quantum yield of H2TPP (ΦΔ = 0.55 0.11) as the
reference standard,21,26 the relative ΦΔ of Rh-SiPc-Rh (2) and
Rh-SiPc (3) were estimated to be 0.48 0.11 and 0.25 0.11,
Figure 4. Normalized excitation spectrum (black, monitored at 750 nm) and fluorescence spectra (blue, λexc = 515 nm and red, λexc = 640 nm) of
2 (a) and 3 (b) in CH2Cl2 (∼1.0 × 10−6 M). Note that the emissions from the Pc moiety of 2 and 3 excited at 515 and 640 nm coincide almost
completely.
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Figure 5. Near-IR phosphorescence spectra of the 1O2 generated from the following photosensitizers: (a) H2TPP (black), Rh-SiPc-Rh (2, blue), and
Rh-SiPc (3, red) excited at 549 nm in CHCl3; (b) ZnPc (black), Rh-SiPc-Rh (2, red), C7-SiPc-C7 (4, blue), and Rh-SiPc (3, green) excited at 666
nm in toluene. Note that the concentrations of the photosensitizers used are different between a and b.
respectively, when excited at the Rh B absorption maximum of
549 nm. Since rhodamine has very low 1O2 production
capability,37 the substantial O2 generation observed must be
1
the result of an energy transfer from the photoexcited Rh B to
1
the Pc moiety which produced the O2 observed in 2 and 3.
To evaluate the 1O2 production capability of the SiPc moiety
1
alone, the O2 phosphorescence spectrum of C7-SiPc-C7 (4),
soluble in toluene, upon photoexcitation at 549 nm was mea-
sured and is shown in Figure 5b, together with those spectra
obtained from ZnPc, 2 and 3, measured under identical con-
ditions. The relative ΦΔ estimated for ZnPc, Rh-SiPc-Rh (2),
Rh-SiPc (3), and C7-SiPc-C7 (4) in toluene were 0.58 0.11,
0.50 0.10, 0.23 0.07, and 0.38 0.08, respectively. These
measurements show that (1) SiPc is quite capable of producing
singlet oxygen even when excited at a nonoptimal wavelength
(e.g., 4 with ΦΔ = 0.38 at 549 nm), (2) the ligation of two Rh B
1
to SiPc increased its O2 quantum yield (2 with ΦΔ = 0.50),
and (3) the presence of an additional Rh B chromophore in 2
resulted in a 2-fold increase in the 1O2 quantum yield of 3 (ΦΔ =
0.23). Thus, while the introduction of the Rh moiety to SiPc is
mainly for its mitochondria-targeting function in cells (vide
1
infra), its presence also allows for O2 generation from Pc’s at
shorter wavelengths that support the Rh-to-Pc energy transfer.
Two-Photon Absorption Properties of 2, 3, and 4.
1
Recent studies showed that O2 can also be generated via two-
photon excitation of a sensitizer.38 In this approach, a chromo-
phore such as Rh B which absorbs at λ < 650 nm, that is, in the
Figure 6. Open-aperture Z-scan traces of 0.1 mM of Rh-SiPc-Rh, (2,
top panel) and Rh-SiPc, (3, bottom panel) excited at 850 nm. The
tissue nontransparent spectral region, by one-photon absorp-
tion, can be excited by two-photon absorption (2PA) at λ > 650
nm, which allows much better depth penetration through
tissues. Other advantages of 2PA-PDT include (1) high-reso-
lution (femtoliter scale) targeting with pinpoint accuracy, thus
minimizing photodamage to tissues along the beam path of a
one-photon PDT, (2) negligible photobleaching of the sen-
sitizing chromophore, and (3) minimal endogenous autofluor-
escence.39,40
To assess the potential of 2 and 3 as 2PA-PDT agents, we
measured their two-photon absorption cross sections, σ2, by
the open aperture Z-scan method.24 The results are shown in
Figure 6, from which the 2PA cross sections of 2 and 3 were
derived to be 1494 GM and 706 GM (where GM = 10−50 cm4 s
photon−1), respectively, at 850 nm. Compared to the σ2 of Rh
B, that is, ∼120 GM at 850 nm,42 an apparent 6-fold enhan-
2PA cross sections determined for 2 and 3 were 1494 GM and 706
GM, respectively.
cement in σ2 for each Rh B chromophore can be seen in these
Rh-SiPc conjugates. To ascertain that this enhancement of σ2 in
2 and 3 is derived principally from Rh B being ligated to SiPc
and not from the SiPc moiety itself, we compared these σ2
values to that of C7-SiPc-C7, 4, which was measured to be 318
GM (data shown in Supporting Information Figure S11). This
comparison clearly shows that the enhancement of σ2 in 2
and 3 is due to the ligation of Rh B to SiPc. This notion is
corroborated by the 2-fold higher σ2 value in 2, with two ligated
Rh B, as compared to that in 1, with only one ligated Rh B.
Such enhancement in two-photon absorption efficiency in 2
and 3 might be due to a possible intramolecular electron transfer
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between Rh B and SiPc, particularly considering the short poly-
ethylene glycol linkage between them. A more detailed photo-
physical study using transient absorption spectroscopy should
shed more light on this issue. Nonetheless, the substantial σ2
measured for 2 and 3 suggests that these conjugates possess
potent 2PA-PDT activity.42
Subcellular Localization of 2 and 3. To ascertain the
mitochondria-localizing ability of Rh-SiPc-Rh (2) and Rh-SiPc
(3), the human nasopharyngeal carcinoma cells (HK-1) were
costained with mitochondria-specific probe. Judging from the
overlapping of the fluorescence signals (yellow dots) emitted
by these Rh-SiPc conjugates (2 and 3, red dots) and the
mitochondria-specific probe (Mito Tracker Green, green dots),
both 2 (Supporting Information Figure S11c, upper panel) and
3 (Supporting Information Figure S11d, upper panel) were
found to localize in the mitochondria of HK-1 cells. The
localization of 2 and 3 in mitochondria was further confirmed
by analyzing the fluorescence-intensity profile drawn across
the cells. The fluorescence profiles of these Rh-SiPc conjugates
along the arrow (Supporting Information Figures S11c and
S11d, lower panels) are similar to the fluorescence profile of the
mitochondria-specific probe. Furthermore, the patterns of colo-
calization of 2, 3, and Rh B (Supporting Information Figure S11a,
upper panel) with the mitochondria-specific probe are very similar.
In contrast, mitochondrial localization of C7-SiPc-C7 (Supporting
Information Figure S11b, upper panel), which possesses no Rh
moiety, was not observed. These observations indicate that Rh B
functioned as a carrier for phthalocyanine to gain entry into the
mitochondria. The mitochondria-localizing capability of 2 and 3
was also demonstrated in a noncancerous cell line, namely, human
umbilical vein endothelial cells (HUVEC), which is shown in
Supporting Information Figure S12.
Figure 7. Photocytotoxicity of Rh B and Rh-SiPc conjugates in HK-1
cells. HK-1 cells (2 × 104 cells/well) were incubated in wells of 96-well
plate for overnight. The cells were then treated with 100 nM of Rh B,
C7-SiPc-C7 (4), Rh-SiPc (3), or Rh-SiPc-Rh (2) for 6 h in dark,
followed by photoirradiation (1−4 J/cm2) and MTT cytotoxicity
assay. Results were expressed as the mean SD of three independent
experiments.
(dark and light).43 Further studies on the cellular uptake of
these conjugates will be conducted to address this issue.
With the establishment of mitochondria-localizing and
photocytotoxic properties of these Rh-SiPc conjugates, the
next logical question is the mode of cell death (i.e., apoptosis vs.
necrosis) induced by these conjugates. To determine the
purported apoptotic cell death associated with the mitochon-
dria-localizing property of Rh-SiPc conjugates, both nuclei
staining method and flow cytometric DNA content analysis
were performed on the HK-1 cells after PDT with 2. No apoptotic
nuclei formation was observed under the light dose 1 J/cm2.
However, apoptotic nuclei were observed for 3 h after PDT
with light doses of 2 and 4 J/cm2 (Supporting Information
Figure S16).
The localizing properties of 2 and 4 in other subcellular
organelles, namely, lysosomes, Golgi bodies and endoplasmic
reticulum (ER), were also studied using the corresponding
organelle-specific probes. The confocal microscopic images
obtained from these colocalization experiments are shown in
Supporting Information Figures S13, S14, and S15. No
significant localization of 2 in lysosomes, Golgi bodies and
endoplasmic reticulum in the HK-1 cells was seen (Supporting
Information Figure S13). As for the C7-SiPc-C7 control (4), no
significant organelle-specific localization in lysosomes, Golgi
bodies and endoplasmic reticulum was detected (Supporting
Information Figures S14 and S15). These observations, taken
together, indicate an almost exclusive subcellular localization of
the Rh-SiPc conjugate, 2, in the mitochondria of these cells.
Photocytotoxicity of 2, 3 and 4 Toward HK-1 cells.
The dark and photo-cytototoxicity of 2 and 3 were measured,
together with Rh B and C7-SiPc-C7 (4), which served as the
reference. The results are shown in Figure 7. With light doses
from 1 to 4 J/cm2, 2, 3, and 4 exhibited a dose-dependent
cytotoxicity to the HK-1 cells. However, photocytotoxicity was
not observed in the Rh B-treated HK-1 cells. In addition, no
significant cytotoxicity (i.e., <10%) due to 2 and 3 was observed
when the HK-1 cells were incubated with these Rh-SiPc conjugates
without photoirradiation. Furthermore, the observed photo-
cytotoxicity of 2, 3, and 4 does not correlate with the measured
1O2 quantum yields (2 > 4 > 3) of these compounds, sug-
gesting that their subcellular localization might exert a crucial
influence on their photocytotoxicity. However, recently Saha
et al showed that the amount of complexes taken up by the
cells is also a key factor in determining their in vitro toxicities
The extent of apoptosis was further evaluated using flow
cytometry and the results are shown in Supporting Information
Figure S17. 2-PDT increased the percentage of sub-G1 cells
from 2% (control) to 10.9% (2 J/cm2) to 20% (4 J/cm2),
indicating that 2-PDT dose-dependently induced tumor cell
apoptosis. Under the light dose of 1 J/cm2, 2-PDT was found
to increase the percentage of G1 cells from 50.8% in the cell
control group to 81.8% after PDT. This observation suggests
that a low dose of 2-PDT would induce G1 cell cycle arrest.
Two-Photon-Induced Cytotoxicity of 2, 3, and 4
Toward Human Cervical Carcinoma (HeLa) and Human
Nasopharyngeal Carcinoma (HK-1) Cells. HK-1 cells were
incubated with 0.5 μM of Rh-SiPc-Rh (2), Rh-SiPc (3), or C7-
SiPc-C7 (4) containing no Rh B moiety, for 6 h. The cells were
then excited at 850 nm, where Rh B and its SiPc conjugates, 2
and 3, showed a two-photon absorption cross-section of ∼120
GM,42 1494 GM and 706 GM, respectively. The confocal
images were captured at one laser shot per minute for a total
of 30 min. Figure 8 shows the confocal images of the cells at
two-photon laser irradiation time t = 0, 5, 25, and 30 min.
Bright fluorescent images (i.e., images captured at t = 0 min)
were seen in cells treated with Rh-SiPc-Rh (2). The fluo-
rescent intensity of the treated cells was time-dependently
decreased. Similar changes in fluorescent intensity were also
seen in cells treated with Rh-SiPc (3). There are two possible
explanations for the reduced fluorescent intensity in cells
after two-photon irradiation, namely, the photobleaching and
the leakage of the Rh-SiPc conjugates from the damaged
cell membrane after PDT. Photobleaching of the Rh-SiPc
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Figure 8. Confocal microscopic images of HK-1 cells treated with 0.5 μM of Rh-SiPc-Rh, (2, top), Rh-SiPc (3, middle), and C7-SiPc-C7 (4, bottom)
obtained at various time points (0, 5, 25, and 30 min) under continuous laser flash excitation at 850 nm (one laser shot per min). The cells were
incubated with 2, 3, and 4 for 6 h before the laser flash excitation.
conjugates is unlikely an explanation as photobleaching of
chromophores resulting from two-photon excitation is
considered negligible.39−41 Leakage of the Rh-SiPc conjugates
through the damaged membrane might be the possible
explanation for the reduced fluorescent intensity of the
stained cell. This explanation is supported by the fact that 2-
or 3-PDT treated cells tend to round up, an early sign of cell
death, after 2PA-PDT treatment. In contrast to Rh-SiPc and
Rh-SiPc-Rh, the morphology of the 4-PDT treated HeLa cells
is similar to the cell image captured at before laser irradiation
(i.e., t = 0 min). These observations clearly showed that 2 and
3 possess 2PA-PDT activities when excited at 850 nm whereas
4, which bear no Rh B chromophore, displayed no significant
2PA-PDT activity. This experiment, however, precludes a
quantitative comparison of the PDT activities of 2 and 3 re-
sulted from one- versus two-photon excitation because the
experimental conditions used (e.g., concentrations of the con-
jugates, cell type, etc.) were quite different. Nonetheless, the
result does demonstrate substantial PDT activities of 2 and 3
induced via two-photon excitation of the Rh B chromophore
potentially. In addition, Figure 8 also shows that 2 gave a
stronger in vitro emission than 3 under two-photon excitation
at 850 nm. This observation is consistent with the 2-fold
higher σ2 value measured for 2 than 3 (Figure 6).
ASSOCIATED CONTENT
* Supporting Information
NMR spectra, high-resolution MALDI-TOF mass spectra, in
vitro imaging, and cytotoxicity results of the compounds synthesized.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: dkwong@hkbu.edu.hk (D.W.J.); wkwong@hkbu.edu.
hk (W.-K.W); nkmak@hkbu.edu.hk (N.-K.M.).
■
Author Contributions
Zhao, Chan, and Li made equal contribution as cofirst author
ACKNOWLEDGMENTS
■
The work described in this paper was partially supported by a
grant from the Research Grants Council of the Hong Kong
SAR, P.R. China (HKBU 202509 and HKBU 202210) and a
grant from the Hong Kong Baptist University (FRG2/09-10/58).
W.-Y. Wong and W.-K. Wong also thanks a grant from Areas of
Excellence Scheme, University Grants Committee of HKSAR,
China (Project No. [AoE/P-03/08]).
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CONCLUDING REMARKS
■
Two axially ligated rhodamine-Si(IV)-phthalocyanine conjugates,
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